The goal of the proposed research is to determine the biochemical mechanism of transfer RNA splicing in higher plant chloroplasts. Transfer RNA genes with 0.4-1.0 kbp introns, or intervening sequences, in their anticodon regions have been described for maize, tobacco, spinach and pea chloroplast genomes. Since these genes are expressed, the intervening sequence RNA must be spliced out of the primary transcript to produce functional tRNAs. The specific aims of this project are: (1) To obtain unspliced precursor transcripts of intron-containing chloroplast tRNA genes by intro transcription, and to characterize these precursor tRNAs by RNA fingerprinting methods. (2) To establish conditions for accurate splicing of these pre-tRNAs, and to characterize the requirements for intervening sequence excision and for exon and intron, and of the splice junction. (4) To partially purify the activity(ies) or factor(s) required for splicing in vitro and to determine their mechanism of action in splicing. Consequently, to determine whether different factors are required to splice different tRNA precursors (e.g., whether different endonucleases excise intervening sequences located at different positions in the anticodon loop). (5) To determine the secondary structures of these introns and to begin to investigate whether structures within the intron or the tRNA are required for excision or ligation. RNA splicing is a fundamental step in the expression of eukaryotic nuclear and organnellar genes. Chloroplast tRNAs, moreover, are an ideal model system for studies on organellar RNA splicing. The chloroplast tRNA introns may exhibit structural homology with a proposed class of fungal and plant mitochondrial mRNA introns. Each pre-tRNA, however, has only a single intron, and gives rise to a simplest system for biochemical investigation of the metabolism of a family of organellar introns. By comparing the mechanism of chloroplast tRNA splicing with those of other organisms, we hope to gain insight to the evolution of splicing mechanisms.